Project Summary Restoration of the numbers and functionality of Beta-cells are anticipated to serve as cures for Type-1 and Type-2 diabetes, respectively. Proof-of-principle has been suggested by results obtained through pancreatic islet transplantation. Although islet transplantation is not lasting and accompanied by significant side effects, the fact that diabetic recipients become insulin-independent suggests that diabetes can be cured by replenishment with functional Beta-cells. Nonetheless, lack of a reliable and functional source of Beta-cells has limited this approach. Recent efforts have focused on increasing total Beta-cell mass by deciphering and manipulating the mechanisms involved in their differentiation, proliferation and regeneration. The Hippo pathway is a kinase pathway responsible for maintaining proper organ size through its negative effect on Yap, a transcriptional co-activator responsible for stimulating cell proliferation and repressing apoptosis. In order to investigate the function of Hippo signaling within the pancreas, immunohistochemical staining for different components of the pathway was completed. Using a phosphorylation-specific Mst1/2 antibody, we have identified Hippo pathway hyperactivity specifically in pancreatic islets. Furthermore, the absence of detectible Yap expression suggests that activation of Hippo signaling is maintaining islets in a quiescent state as demonstrated by the low mitotic index of these structures. Pancreas-specific mouse knockouts have confirmed the requirement for Hippo signaling in the development of the pancreas. Specifically, loss of Mst1/2 led to a progenitor phenotype while loss of Yap led to a decreased number of viable offspring as well as a decrease in total pancreas mass. Both crosses led to significantly increased blood glucose concentrations. We propose to use conditional mouse models with islet-targeted deletion of Mst1/2 and Yap in order to investigate the role of Hippo signaling on normal pancreas and Beta-cell development. Furthermore, an inducible knockout system will be used to determine the effect of Hippo signaling on maintenance of Beta-cells within the adult mouse. The second aim will assess the role of Hippo signaling during Beta-cell regeneration. Using an inducible model of Diphtheria toxin-mediated Beta-cell destruction, we will test the effect of Yap knockout on Beta-cell recovery. Furthermore, gene expression analysis will be used to determine which genes are differentially regulated, both in the presence or absence of Yap, during Beta-cell regeneration. Lastly, the third aim will determine the role of Hippo signaling in maintenance and expansion of human Beta-cells intended for transplantation purposes. RNA interference will be used to decrease the expression of Hippo pathway components within cultured human islets; the effect of which on Beta-cell proliferation and survival will then be determined using in vitro and in vivo systems.